Sensor

ABSTRACT

A sequencer that measures a nucleic acid sequence in a nucleic acid strand includes: a base material having a surface made of silicon, and a fibrous protrusion that is made of silicon dioxide and is directly joined to the surface of the base material made of silicon, wherein a plurality of the nucleic acid strands are fixed onto the fibrous protrusion.

BACKGROUND

1. Technical Field

The present invention relates to a sequencer that measures a sequence ofnucleic acids such as DNAs.

2. Background Art

As a conventional sensor, there is a DNA sensor, for example, as shownin FIG. 20. The DNA sensor includes: a substrate 1; and functionalmolecules, that is, DNA probes 2, which are fixed onto the substrate 1.The DNA probes 2 form double strands together with complementary DNAs 3as ligand molecules. Subject materials such as blood, saliva and riverwater, which have a possibility of containing a variety of DNAs 3, areexposed to the DNA probes 2, and thereafter, it is sensed whether or notthe double strands are formed, whereby it can be detected whether or notthe DNAs 3 of a sensing target are present in these subject materials.

As an analysis method, for example, there is a method of detecting adouble strand forming region of the DNA probes 2 and the complementaryDNAs 3 in such a manner that the varieties of DNAs 3 to be exposed aremarked in advance with a fluorescent substance 4, and each of the DNAs 3is reacted with each of the above DNA probes 2, and thereafter,fluorescence of this reaction region is measured.

Examples resembling the DNA sensors as described above are disclosed inJapanese Patent Laid-open Publication No. H4-505763 and Japanese PatentLaid-Open Publication No. 2007-285927.

Another example regarding a sequencer that measures a sequence (basesequence of a nucleic acid such as DNA is disclosed in Japanese PatentLaid-open Publication No. 2002-525125.

SUMMARY OF THE INVENTION

In the conventional sensor, sensitivity thereof has sometimes beenlowered. The reason for this is because a formation density of thefunctional molecules has been low. Specifically, heretofore, it has beendifficult to fix a sufficient amount of the functional molecules (DNAprobes 2) to a predetermined region, and hence, an amount of signalsemitted by a combination of the functional molecules 2 and the ligandmolecules (DNAs 3) has also been small. As a result, an amount of thesignals sensed by the sensor has also been reduced, and the sensitivityof the sensor has been lowered.

In this connection, it is an object of the present invention to providea sequencer that enhances sensitivity thereof.

A sensor according to the present invention includes:

a base material having a surface made of silicon:

a plurality of fibrous protrusions which are made of silicon dioxide andare directly joined to the surface of the base material made of silicon:and

a plurality of functional molecules respectively formed on the fibrousprotrusions.

Moreover, a sequencer according to the present invention includes:

a base material having a surface made of silicon;

a fibrous protrusion that is made of silicon dioxide and is directlyjoined to the surface of the base material made of silicon,

wherein plurality of nucleic acid strands are ixed onto the fibrousprotrusion, and nucleic acid sequences of the nucleic acid strands aremeasured.

In accordance with the sensor according to the present invention,sensitivity thereof can be enhanced. The reason for this is becausefunctional molecules can be fixed onto a base material with a highdensity. Specifically, in accordance with the present invention n, asurface area of the base material is increased by the above-mentionedfibrous protrusions, and the functional molecules can be formed with ahigh density even in a narrow space. Hence, reaction between therespective functional molecules and the ligand molecules can be obtainedsufficiently, and can be detected as a large signal. As a result, thesensitivity of the sensor can be enhanced.

Moreover, in accordance with the sequencer according to the presentinvention, measurement accuracy of nucleic acid sequences can beenhanced. The reason for this is because a density of the nucleic acidsin a unit region can be increased. Specifically, in accordance with thepresent invention, a plurality of fibrous protrusions are formed on thesubstrate, and the nucleic acids are amplified on the fibrousprotrusions. Accordingly, a surface area of a support body is increased,and an amplified amount of the nucleic acids in one region is increased.Then, as a result, a signal detectable from the whole of the nucleicacid colony is increased. As a result, the measurement accuracy of thenucleic acid sequences can be enhanced.

BRIEF DESCRIPTION CF THE DRAWINGS

The present invention will become readily understood from the followingdescription of preferred embodiments thereof made with reference to theaccompanying drawings, in which like parts are designated by likereference numeral and in which:

FIG. 1 is a crass-sectional view of a sensor according to a firstembodiment of the present invention;

FIG. 2 is an enlarged cross-sectional view of a main portion of thesensor;

FIG. 3 is a SEM picture of fibrous protrusions of the sensor;

FIG. 4 is a cross-sectional view for describing a manufacturing methodof the sensor;

FIG. 5 is a cross sectional view for describing the manufacturing methodof of the sensor;

FIG. 6 is a cross-sectional view for describing the manufacturing methodof the sensor;

FIG. 7 is a cross-sectional view for describing the manufacturing methodof the sensor;

FIG. 8 is a crass-sectional view for schematically describing themanufacturing method of the sensor;

FIG. 9 is a cross-sectional view for schematically describingmanufacturing method of the sensor;

FIG. 10 is a cross-sectional view for schematically describing themanufacturing method of the sensor;

FIG. 11 is a cross-sectional view for schematically describing themanufacturing method of the sensor;

FIG. 12 is a cross-sectional view for schematically describing themanufacturing method of the sensor;

FIGS. 13A to 13C are cross-sectional views schematically showing a mainportion of the sensor;

FIG. 14 is a graph showing a result of X-ray spectroscopic analysis forthe fibrous protrusions in the first embodiment of the presentinvention;

FIG. 15 is a cross-sectional view of a sensor according to a secondembodiment of the present invention;

FIG. 16 is a cross-sectional view of a sequencer according to a thirdembodiment of the present invention;

FIGS. 17A to 17F are views showing a process of amplifying nucleic acidsby using the sequencer according to the third embodiment;

FIGS. 18A and 18B are views showing a process of measuring sequences ofthe nucleic acids by using the sequencer according to the thirdembodiment;

FIG. 19 is a cross-sectional view of a sequencer according to a fourthembodiment of the present invention; and

FIG. 20 is a cross-sectional view of a conventional DNA sensor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(First Embodiment)

In a first embodiment, a description will be made of a DNA sensor takenas an example of an embodiment of the present invention.

As shown in a cross-sectional view of FIG. 1, the DNA sensor has asubstrate 5 made of silicon as a base material. A surface of thesubstrate 5 is partitioned into a plurality of regions 6 to 8, and foreach of these regions 6 to 8, a plurality of fibrous protrusions 9 madeof silicon dioxide, in each of which one end of the fibrous protrusion 9is directly joined to the substrate 5, are formed. Herein, “directlyjoined” refers to a state where the fibrous protrusions 9 are directlyformed on the substrate 5, and atoms or molecules as being componentsfor constituting the fibrous protrusions 9, are directly bonded to thesubstrate 5, and generally, refers to a state where the molecules arecovalently bonded to each other. In the first embodiment, silicon atomson the surface of the substrate 5 and silicon atoms in the fibrousprotrusions 9 are covalently bonded to each other through oxygen atomsin the atmosphere. Moreover, a silane coupling agent is adhered ontosurfaces of these fibrous protrusions 9, whereby coating layers (notshown) in a matrix shape are formed. Moreover, in the first embodiment,linker molecules (not shown) are contained in these coating layers, andas shown in FIG. 2, a plurality of DNA probes 10 are joined by usingthese linker molecules as bases.

Note that, in the first embodiment, functional molecules, that is, DNAprobes 10 different for each of the regions 6 to 8 on the siliconsubstrate 5 are joined thereto. Specifically, the DNA probes 10 arepolymers in which monomers of adenine (A), guanine (G), thymine (T) andcytosine (C) are arrayed in a variety of orders, and in the firstembodiment, the DNA probes 10 are formed, in which such order sequencesare changed for each of the regions 6 to 8.

In the first embodiment, a silicon substrate 5 made of single crystalsilicon is used as the substrate 5. However, the surface thereof justneeds to be made of silicon, and the silicon may be in monocrystailine,polycrystalline, amorphous, and other states. Moreover, a so-calledSilicon On Insulator (SOI) substrate may be used, for example, in whichfront and back surfaces are made of silicon, and a silicon dioxide layeris inserted therebetween. Moreover, as the substrate 5 of theembodiment, a substrate 5 with a thickness of approximately 400 μm isused. It is suitable that the thickness of the substrate 5 be 1 mm orless.

Moreover, in the first embodiment, an overall length of the fibrousprotrusions 9 is set at 1 to 500 μm, a diameter thereof is set at 0.01μm to 10 μm, and an interval among the plurality of fibrous protrusions9 is set at 0.01 μm to 10 μm.

As shown in FIG. 3, the fibrous protrusions 9 are grown until minutelyundulating and curling in order to increase a surface area thereof, eachone of the fibrous protrusions 9 has a crimped shape, and the fibrousprotrusions 9 are densely formed in a state of intertwining with oneanother. Moreover, those branched in free directions may be mixedlypresent in the fibrous protrusions 9. The fibrous protrusions 9intertwine with one another, and have a plurality of such branches,whereby the fibrous protrusions 9 are robustly formed.

Moreover, the fibrous protrusions 9 of the first embodiment are de ofamorphous silicon dioxide, and thereby have a structure that is hard tobreak as compared with single crystal silicon dioxide. Note that, when aregion in which the fibrous protrusions 9 are formed is measured byX-ray spectroscopic analysis, then as shown in FIG. 14, there are nolarge peaks except for a peak of Si (110), which is located at a regionwhere 2θ/degree is approximately equal to 47° and it is concluded thatthe fibrous protrusions 9 are made of the amorphous silicon dioxide.

In the first embodiment, in order to further facilitate the bondingbetween the surfaces of the fibrous protrusions 9 and the DNA probes 10,the silane coupling agent containing the linker molecules adhered ontothe surfaces of the fibrous protrusions 9. However, these silanecoupling agent and linker molecules are not essential compositions.

Note that the silane coupling agent enhances bonding properties amongthe surfaces of the fibrous protrusions 9, and an agent such aspoly-L-lysine, which has a composition other than that of the silanecoupling agent, may be used as such an agent.

Moreover, the linker molecules serve as the bases of the DNA probes 10,and for example, there are mentioned aryl acetylene, ethylene glycololigomer, diamine, amino acid and the like, and mixtures thereof. Notethat, in some cases, the above-mentioned silane coupling agent itselfbecomes the linker molecules.

Next, a manufacturing method of the DNA sensor of the first embodimentwill be described.

(a) First, as shown in FIG. 4, the substrate 5 is partitioned intoregions, and boundary portions among these regions are covered with aprotection film 11 made of silicon dioxide, or resin of photoresist etal. In FIGS. 4 to 7, any one of the regions 6 to 8 shown in FIG. 1 isonly shown.

(b) Next, when at least any one of gases of CF₄, CHF₃, C₂F₆, C₃F₈, andC₄F₈; is decomposed in plasma, and is introduced onto the surface of thesilicon substrate 5, then as shown in FIG. 5, a seed layer 12 is formedon the surface of the substrate 5.

The seed layer 12 is a layer made of an organic polymer containingcarbon element referred as C and fluorine element referred as F or C, F,and H elements, and the seed layer 12 can be formed by decomposingfluorocarbon-series gas such as the above-mentioned CF₄, CHF₃, C₂F₆,C₃F₈, and C₄F₈ by using a plasma CVD method.

In the case of using inductive coupled plasma (ICP) in order todecompose the above gas in the plasma, a decomposition degree of the gasis increased, and it is easy to uniformly form the seed layer 12.

Note that, in order to uniformly form the fibrous protrusions 9 insubsequent steps by the seed layer 12, the surface of the siliconsubstrate 5 is desirably made of pure silicon. However, the surface ofthe silicon substrate 5 may be in a state where an extremely thinnatural oxide film is formed thereon.

(c) Next, as shown in FIG. 6, the protection film ii is removed bychemical treatment using a chemical agent. As in the first embodiment,the seed layer 12 formed by the plasma CVD method has a relativelystrong chemical tolerance, and accordingly, only the protection film 11can be selectively removed.

(d) Thereafter, the substrate 5 is fired at 1000 to 1150° C. under alow-oxygen atmosphere. Then, as shown in FIG. 7, on the surface (regionwhere the seed layer 12 of FIG. 5 is formed) of the substrate 5, thefibrous protrusions 9 made of silicon dioxide are formed. In accordancewith this method, the fibrous protrusions 9 turn to a state of beingdirectly joined to the silicon surface of the substrate 5, whereby thefibrous protrusions 9 are excellent in heat durability, and become lesslikely to be peeled off. Moreover, the fibrous protrusions 9 are formedin such a manner that the substrate 5 grows around the seed layer 12taken as cores, and accordingly, other substances are less likely to bemixed thereinto, and a composition with fewer impurities is achieved.

Note that, in the firing step, the fibrous protrusions 9 are not formedon the surface of the silicon substrate 5, on which the seed layer 12 isnot formed.

In the firing step, it is conceived that the seed layer 12 made of therespective atoms of C and F or the respective atoms of C, F, and Hdisappears by being burnt, and does not become a factor to inhibithydrophilicity.

It is also possible to form the fibrous protrusions 9 by using a metalcatalyst. In such a case, first, a metal catalyst layer is formed on thesurface of the substrate 5 by evaporation, sputtering, and the like. Asthe metal to be used as the catalyst. Au, Fe, Ni, Co, and the like aredesirable, and a thickness of the metal catalyst layer is approximatelyseveral nanometers.

Next, when the substrate 5 on which the metal catalyst layer is formedis fired at 1000 to 1150° C. under the low-oxygen atmosphere, thefibrous protrusions 9 made of silicon dioxide are formed on the surfaceof the substrate 5. Also in this case, the fibrous protrusions 9 turn tothe state of being directly joined to the silicon surface of thesubstrate 5, whereby the fibrous protrusions 9 are excellent in heatdurability, and become less likely to be peeled off. Moreover, thefibrous protrusions 9 are formed in such a manner that the substrate 5grows around the seed layer 12 taken as cores, and accordingly, othersubstances are less likely to be mixed thereinto, and the compositionwith fewer impurities is achieved.

Note that the low-oxygen atmosphere is composed of an atmosphere ofnitrogen, argon, or the like, or of a vacuum atmosphere.

In the case where the fibrous protrusions 9 are formed by using themetal catalyst by the above method, the metal catalyst may be supportedon the fibrous protrusions 9.

Next, a method of bonding the fibrous protrusions 9 and the DNA probes(denoted by reference numeral 10 in FIG. 2) to each other will bedescribed with reference to FIGS. 8 to 13. FIG. 8 to FIG. 13schematically show the respective components while changing scalesthereof in order to facilitate the description.

(a) As shown in FIG. 8, the surface of the substrate 5 is coated with acoating layer 13 containing the silane coupling agent. To the coatinglayer 13, the linker molecules (not shown) in which reaction groups aremodified by blocking groups are bonded. Specifically, in the firstembodiment, the above-mentioned blocking groups are bonded to thesurfaces of the fibrous protrusions 9 through the linker molecules andthe coating layer 3. In the first embodiment, a substance capable ofunblocking upon reaction with light is used as the blocking groups.Examples of the blocking groups include ortho-nitrobenzyl derivative,6-nitroveratryl oxycarbonyl, 2-nitrobenzyl oxycarbonyl, cinnamoylderivative, and the like. In the case where the linker molecules are notused, the blocking groups and the reaction groups of the fibrousprotrusions 9 just need to be bonded to each other

(b) Next, as shown in FIG. 9, the surface of the substrate 5 is maskedby a mask 14 so as to expose only the regions 7 and 8 as a part amongthe plurality of regions 6 to 8 on the substrate 5.

(c) Subsequently, when the surface of the substrate 5 is irradiated withlight from above the mask 14, the blocking groups are removed(unblocked), and the reaction groups of the linker molecules areexposed. The linker molecules in the coating layer 13A shown in FIG. 9are in a state where the blocking groups are removed and the reactiongroups are exposed. In this case, as irradiation light, light with apredetermined wavelength, which is capable of unblocking the blockinggroups, is used.

(d) Thereafter, when a solution containing a nucleotide monomer 15 ofany of adenine (A), guanine (G), thymine (T), and cytosine (C) isinjected onto the silicon substrate 5, then as shown in FIG. 10, thereaction groups of the exposed linker molecules and the monomer 15 arebonded to each other.

In the first embodiment, the blocking groups and the fibrous protrusions9 are bonded to each other through the linker molecules. However, in thecase where the blocking groups are directly bonded to the fibrousprotrusions 9 without using the linker molecules, the monomer 15 and thereaction groups of the fibrous protrusions 9 in which the blockinggroups are unblocked are bonded to each other.

Herein, in the first embodiment, a monomer modified by photoreactiveblocking groups is used as the monomer 15. These blocking groups may bethe same substance as the blocking groups of the linker molecules, ormay be a different substance therefrom.

Next, as shown in FIG. 11, the surface of the substrate 5 is masked byanother mask 16, whereby only such partial regions 6 and 7 are exposed.The regions 6 and 7 thus exposed may be the same region as the region towhich the monomer 15 is bonded in the previous step. Then, the regions 6and 7 are irradiated with light with a predetermined wavelength, wherebythe blocking groups in the monomer on the region 7 and in the linkermolecules of the coating layer on the region 6 are respectivelyunblocked. FIG. 11 shows the coating layer 13A and the monomer 15A, fromwhich the blocking groups are unblocked

(f) Then, as shown in FIG. 12, in the same manner as in the step offixing the monomer 15 described above, a solution of a monomer 17modified by the blocking groups is injected, and the monomer 15A inwhich the blocking groups on the region 7 are unblocked and the linkermolecules in which the blocking groups on the region 6 are unblocked arebonded to each other.

As described above, when the step of bonding four types of thenucleotide monomers to one another by using different masks is repeated,the DNA probes 10 different in type can be formed for each of theregions 6 to 8.

FIG. 13A, FIG. 13B, and FIG. 13C schematically show the DNA probes 10,which are formed on the surfaces of the fibrous protrusions 9 on theregion 6, the region 7, and the region 8, respectively. The number ofnucleotide monomers which form each of the DNA probes 10 is notparticularly limited; however, approximately 10 to 30 (length:approximately 0.003 to 0.01 μm) is suitable for forming the doublestrand. Moreover, by a combination of the masks, DNA probes 10 havingvarious types of sequences can also be formed quickly.

Moreover, in the first embodiment, the photoreactive blocking groups areused as the blocking groups; however, for example, substances capable ofunblocking by a current, an electrolyte, an ion beam, and the like maybe used. In this case, in the step of unblocking the blocking groups,the current, the electrolyte, and the on beam just need to beindividually applied.

In the first embodiment, the method of polymerizing the monomer bycontrolling the reaction regions by using the blocking groups asmentioned above is used. However, for example, a method of stacking themonomers on a predetermined region by using a photolithographytechnology may also be used.

Moreover, in the first embodiment, as an example of a method ofselectively forming the fibrous protrusions 9 on the predeterminedregions 6 to 8 of the substrate 5, there is mentioned the method offorming the protection film (denoted by reference numeral 11 in FIG. 4)in advance on the surface of the substrate; however, other methods maybe selected. For example, there may be selected such a method, in whichthe fibrous protrusions 9 are first formed on the whole of the region onthe silicon substrate 5, the regions 6 to 8 on which the fibrousprotrusions 9 are desired to be left are thereafter covered with theprotection film made of the resin or the like, the fibrous protrusions 9on the regions from which the fibrous protrusions 9 are desired to beremoved are removed by etching by using a usual chemical agent such asHF and BHF, and the protection film is thereafter removed. In this case,the above-mentioned thermal oxide film is not formed on the exposedsurface.

As a method of removing the protection film, it is desirable to removethe protection film by chemical treatment using a solvent and the like.This is because, in the case of using the chemical treatment, the minutefibrous protrusions 9 are less likely to be broken as compared withmechanical treatment.

For example, the DNA sensor formed as described above has double strandsfrom the DNAs marked in advance with a fluorescent substance or the likeand from the DNA probes 10, or the DNA sensor has a fluorescentsubstance inserted into the formed double strands. Thus, the DNA sensorcan measure by means of light whether or not the double strands areformed. Besides, it can be measured whether or not the double strandsare formed based on detecting a change of the current or voltage,surface roughness, and the like as well as the light.

A description will be made below of effects in the first embodiment.

In the first embodiment, sensitivity of the sensor can be enhanced. Thereason for this is because the DNA probes 10 as the functional moleculescan be formed with a high density, Specifically, in the presentinvention, a surface area of the substrate 5 is increased by the fibrousprotrusions 9, and receptors can be formed with a high density even in anarrow space. Hence, the reaction between the respective DNA probes 10and complementary DNAs (ligand molecules) can be obtained sufficiently,and can be detected as a large signal. Then, as a result, thesensitivity of the sensor can be enhanced.

Moreover, in the first embodiment, since the fibrous protrusions 9 aremade of silicon dioxide, the surfaces thereof are hydroxylated more ascompared with the surface of the substrate 5. Hence, bonding propertiesbetween the fibrous protrusions 9 and a chemical substance such as thelinker molecules are enhanced, and the DNA probes 10 can be fixed with ahigher density.

Moreover, as compared with the exposed surface (surface on which thefibrous protrusions 9 are not formed) of the substrate 5, the regionwhere the fibrous protrusions 9 are formed has high hydrophilicity andwater retention properties. Hence, in the case of supplying a polarsolvent onto the substrate 5, an occurrence of bubbles can be suppressedin the region where the DNA probes 10 are formed since the polar solventhas high affinity with the fibrous protrusions 9. Specifically, in thefirst embodiment, the bubbles are less likely to be generated, forexample, in the step of injecting the monomer solution onto thesubstrate 5 or injecting a measurement liquid thereonto. Then, as aresult, the DNA probes 10 can be formed with a higher density, or themeasurement can be performed with high accuracy. Note that, at least oneof the hydrophilicity and the water retention properties contributes toreduction of she bubbles.

Moreover, in the first embodiment, the DNA probes 10 are formed on thefibrous protrusions 9, and accordingly, in the region where the DNAprobes 10 are fixed, reflectivity thereof is lowered as compared withthe flat surface. Hence, for example, in the case of performing thefluorescence analysis, excitation light generated in the case ofirradiating silicon atoms with light is mixed with reflected light fromthe substrate 5, and sometimes becomes a factor of noise at the time ofthe fluorescence detection. However, in the present invention, theabove-described excitation light can be reduced by being shaded by thefibrous protrusions 9, and a cordingly, the noise can be reduced as aresult.

(Second Embodiment)

A main difference between the second embodiment and the first embodimentis a shape of the substrate 5 as shown in FIG. 15. Specifically, in thesecond embodiment, recessed portions 18 are formed in advance on thesurface of the substrate 5 by wet etching or dry etching, and on bottomsurfaces of the recessed portions 18, the fibrous protrusions 9 areformed. In such a way, in the second embodiment, the fibrous protrusions9 on the respective regions 6 to 8 can be suppressed from growing beyondthe respective regions 6 to 8. Specifically, a growing direction of thefibrous protrusions 9 is suppressed by suppressing surface diffusionthereof. Hence, shape accuracy of the regions where the fibrousprotrusions 9 are formed can be enhanced.

Moreover, in the second embodiment, for example, in the step ofinjecting the monomer solution onto the substrate 5 or infecting themeasurement liquid thereonto, the diffusion of the monomer solution orthe measurement liquid can be prevented. Hence, the inspectionsensitivity of the sensor is enhanced.

In the second embodiment, the fibrous protrusions 9 are formed on thebottom surfaces and side wall surfaces of the recessed portions 18;however, the fibrous protrusions 9 may be formed only on the bottomsurfaces of the recessed portions 18, or only on the sidewall surfacesthereof.

A dent depth of the recessed portions 18 is preferably made deeper thana thickness of layers where the fibrous protrusions 9 are formed, sinceit then becomes easy to suppress the diffusion. More preferably, thedent depth of the recessed portions 18 is made deeper, by several tenmicrometers or more, than the thickness of the layers where the fibrousprotrusions 9 are formed. Note that ranges of the recessed portions 18can be appropriately selected in response to the purpose.

Descriptions of other configurations and effects, which are similar tothose of the first embodiment, will not be given.

In the second embodiment, the DNA sensor is taken as an example of thesensor, and the DNA probes are taken as an example of the functionalmolecules; however, RNAs may be used as the functional molecules.Similarly to the DNAs, the RNAs can also be formed on the predeterminedregions easily by using the blocking groups.

(Third Embodiment)

In the third embodiment, a sequencer for measuring a sequence (basesequence) of nucleic acids such as DNAs is described as follows.

As shown in FIG. 16, similarly to the DNA sensor of the firstembodiment, the sequencer of the third embodiment also includes: asubstrate 19 as a base material; and a plurality of fibrous protrusions23 selectively formed on regions 20 to 22 of the substrate 19. In thethird embodiment, an interval (interval at root portions) between aplurality of fibrous protrusions 23 in the respective regions 20 to 22is set at 1 to 10 μm, and a diameter of the protrusions 23 is set at0.01 μm to 10 μm. Moreover, an overall length of the fibrous protrusions23 is approximately 1 to 500 μm.

Similarly to the first embodiment, the fibrous protrusions 23 are grownuntil minutely undulating and curling in order to increase a surfacearea thereof more each one of the fibrous protrusions 23 has a crimpedshape, and the fibrous protrusions 23 are densely formed in a state ofintertwining with one another. Moreover, those protrusions branched infree directions may be mixedly present in the fibrous protrusions 23. Insuch a way, a structure is obtained, in which a stress load is likely tobe dispersed also against an external pressure, and mechanical strengthis strong.

Moreover, similarly to the first embodiment, the fibrous protrusions 23of the third embodiment are made of amorphous silicon dioxide, and havea structure less likely to be broken as compared with single crystalsilicon dioxide.

A manufacturing method of the fibrous protrusions 23 as described aboveis similar to that of the DNA sensor shown in the first embodiment.First, on the substrate 19, fluorocarbon-series gas is decomposed inplasma by using the plasma CVD method, and a seed layer made of anorganic polymer containing C and F elements or C, F, and H elements isformed. The seed layer can be formed by being fired at 1000 to 1150° C.under a low-oxygen atmosphere. It is also possible to form the fibrousprotrusions 23 by using a metal catalyst. Details of the manufacturingmethod are similar to those of the first embodiment, and accordingly, adescription thereof will not be given.

Next, a nucleic acid amplification method using the sequencer of thethird embodiment is described as follows.

(a) First, DNA or RNA (hereinafter, referred to as nucleic acid) forwhich the nucleic acid sequence (base sequence) is desired to bemeasured is randomly split by using enzymes such as EcoRI and HhaI, anda nucleic acid fragment shown in FIG. 17A is formed. Although dependingon reading accuracy in the sequence reading step to be described later,a length of the nucleic acid fragment 24 is usually a length of 10 to4000 base pairs. The nucleic acid fragment 24 may have either form of asingle strand or a double strand.

(b) Next, an adapter 25 containing an oligonucleotide sequence Y1 isbonded to one end of this nucleic acid fragment 24, and an adapter 26containing an oligonucleotide sequence Z1 is bonded to the other end ofthe nucleic acid fragment 24. Thus, a nucleic acid template T1 as shownin FIG. 17A is formed. Note that the oligonucleotide sequence refers toa sequence of a short nucleotide sequence made of some base pairs, andcontains, as constituents, bases of adenine (A), guanine (C), cytosine(C), and thymine (T) or uracil (U).

It is preferable that each of the oligonucleotide sequence Y1 and theoligonucleotide sequence Z1 be formed of approximately 5 to 100nucleotides. In the third embodiment, the oligonucleotide sequences Y1and Z1 are bonded to the most terminal ends of the nucleic acid templateT1. However, a vicinity of the terminal end (preferably, within a rangefrom 0 to 100 nucleotides from a terminal end 5′ or terminal end 3′ ofthe nucleic acid template T1) can be bonded to a support body 1.

(c) Moreover, in the third embodiment, a plurality of colony primers X1and X2 are prepared. The colony primer X1 has a sequence capable ofbeing hybridized with the oligonucleotide sequence Z1, and the colonyprimer X2 has a sequence capable of being hybridized with anoligonucleotide sequence (Z2 to be described later) of a nucleic acidtemplate (T2 to be described later) formed by extension of the colonyprimer X1. Specifically, the colony primer X2 has a sequencecorresponding to the oligonucleotide sequence Y1

(d) Next, in the third embodiment, OH groups of the fibrous protrusion23 of the support body 1 are induced by aminopropyltriethoxysilane (ATS)and the like, and a surface of the fibrous protrusion 23 isfunctionalized by a difunctional coupling agent.

(e) Then, a solution containing the colony primers X1 and X2 and thenucleic acid template T1 is provided to the functionalized surface ofthe fibrous protrusion 23, and as shown in FIG. 17B, the respectiveterminals ends 5′ of the colony primers X1 and X2 and the nucleic acidtemplate T1 are covalently bonded to the surface of the fibrousprotrusion 23. Note that, a diagonally shaded portion of FIG. 17Bindicates the fibrous protrusion 23. In the third embodiment, the colonyprimers X1, X2, and the nucleic acid template T1 are bonded to thesurface of the fibrous protrusion 23 by amide bond; however, as otherbonds, covalent bonds such as ester bond and thiol bond may also beselected as above-mentioned.

(f) Next, as shown in FIG. 17C, the nucleic acid template T1 and thecolony primer X1, which are bonded to the fibrous protrusion 23, areinduced, and the oligonucleotide sequence Z1 (adapter 26) of the nucleicacid template T1 and the colony primer X1 are hybridized with eachother. Inducing conditions at this time may include, for example,putting such hybridization targets at a temperature of approximately 65°C.

(g) Thereafter, as shown in FIG. 17D, under an appropriate temperaturecondition and the presence of nucleic acid polymerase (for example,DNA-dependent DNA polymerase, reverse transcriptase molecules, RNApolymerase, or the like), the nucleic acid is synthesized while using,as base substrates, four types of nucleotide precursors, that is, fourtypes of deoxynucleoside triphosphate, which are: deoxyadenosinetriphosphate (hereinafter, abbreviated as dATP); deoxyguanosinetriphosphate (hereinafter, abbreviated as dGTP); deoxycytidinetriphosphate (hereinafter, abbreviated as dCTP); and deoxythymidinetriphosphate (hereinafter, abbreviated as dTTP).

(h) As described above, the nucleic acid polymerase extends the colonyprimer X1 from the terminal end 3′ thereof by using the nucleic acidtemplate T1 as a template. Then, after such extension of the primer iscompleted, a second nucleic acid strand complementary to the firstnucleic acid template T1, that is, a nucleic acid template T2 iscreated.

(i) Thereafter, for example by heating, a state as shown in FIG. 17E isbrought, where two separate nucleic add strands (nucleic acid templatesT1 and T2) are immobilized on the fibrous protrusion 23.

(j) Then, as shown in FIG. 17F, both of the nucleic acid template T1first immobilized and the nucleic acid strand (nucleic acid template T2)formed by extending the colony primer X1 function as the nucleic acidtemplates. Specifically, the nucleic add template T1 and the nucleicacid temperature T2 are hybridized with another immobilized colonyprimer X1 or colony primer X2 to thereby cause the primer extensionindividually. Then, the nucleic acid is separated to thereby amplify thenucleic acid strands immobilized to the fibrous protrusion 23. Thenucleic acid strands are amplified as described above, whereby a nucleicacid colony is created. Note that, in the third embodiment, the nucleicacid colony includes the one having the sequence corresponding to thefirst nucleic acid template T1, and the one having the sequencecomplementary to the nucleic acid template T1 (that is, corresponding tothe nucleic acid template T2).

As described above, even in the other regions on the support body 1, thenucleic acid colony composed of two types of nucleic acid strands can beformed in a similar way by using, as the templates, the nucleic addssplit randomly. This nucleic acid colony contributes more to theminiaturization by being formed at a small interval of less than 10 μm.

Next, the nucleic acid sequence can be measured after the nucleic acidis amplified.

In the third embodiment, among the two types of nucleic acid strandswhich compose the nucleic acid colony, the measurement is performed forthe sequence of the nucleic acid template T1. At this time, for example,the nucleic acid strand complementary to the nucleic acid template T1just needs to be cut off from the fibrous protrusion 23. Specifically,since the nucleic acid template T2 is bonded to the fibrous protrusion23 through the colony primer X1, the colony primer X1 can be cut offfrom the fibrous protrusion 23 by a restriction enzyme. Accordingly, asshown in FIG. 18A, a nucleic acid colony made only of the nucleic acidstrands corresponding to the nucleic acid template T1 can be formed.

Next, a solution containing: a primer 27 capable of being hybridizedwith the oligonucleotide sequence Z1 on the terminal end 3′ side of thenucleic acid template T1; four types of the modified nucleotideprecursors (A, T, C, and G shown in FIG. 18A); and the nucleic acidpolymerase is flown on the fibrous protrusion 23. At this time, the fourtypes of nucleotide precursors are modified in advance by a variety offluorescent substances so as to emit mutually different types ofexcitation light. Then, at the moment when the nucleotide precursors arehybridized, the fluorescent substances are excited by a laser beam, andemit a fluorescent color. At this time, in one nucleic acid colony, onetype among the four types of nucleotide precursors is bonded, andaccordingly, color emissions with the respective wavelengths can beconfirmed by the nucleic acid colony. Next, as shown in FIG. 18B, thefour types of nucleotide precursors and the enzymes are flown again ontothe fibrous protrusion 23, whereby the next nucleotide precursors areextended from the primer 27, are hybridized with the nucleic acidtemplate T1, and emit colors with unique wavelengths.

As described above, in the process where the primer 27 is extended, andthe nucleic acid sequence complementary to the nucleic acid templates T1is formed from the terminal end 3′ side, the color emissions occur in avariety of orders depending on the nucleotide sequence of the nucleicacid template T1. Hence, the color emissions are recorded, whereby thenucleotide sequence of the nucleic acid templates can be read for eachnucleic acid colony, and a sequence of the respective diffused fragmentsis revealed.

In FIG. 18A described above, if the nucleotide precursors are chemicallymodified in advance so that an extension reaction cannot occursubsequently to an occurrence of one hybridization reaction, then onenucleotide sequence on the single can be reliably determined.Specifically, a chemical modification substance is removed afterward,and the chemically modified nucleotide precursors are flown again,whereby the reactions can be reliably determined one by one.

Here, with regard to the nucleic acid template, if a large number of thenucleic acids for which the sequences are desired to be measured areprepared in advance, and are formed by being split randomly, then notonly the sequence of the nucleic acid fragments but also the sequence ofthe original nucleic acids can be analyzed. In the third embodiment, thesupport body 1 is partitioned in the plurality of regions, the fibrousprotrusions 23 are formed selectively in the regions thus partitioned,and the nucleic acid colony is formed for each of the regions. Hence, asshown in Table 1, the sequences read from the nucleic acid colonies inthe respective regions are compared with one another, and if there arethe same sequence portions, then the portions concerned are arrayed soas to superpose on each other, whereby the entire sequence of theoriginal nucleic acids can be read gradually.

TABLE 1 Measurement Region Read Sequence Region 3 AATCGCTATTTACCCGGRegion 4         TTTACCCGGATTCGCCC Region 5 GAATCGC

A description will be made below of effects in the third embodiment.

In the third embodiment, a density of the nucleic acids in the unitregion is increased, and measurement accuracy of the nucleic acidsequence can be enhanced. The reason for this is because the pluralityof fibrous protrusions 23 are formed on the substrate 19, and thenucleic acids are amplified on the fibrous protrusions 23. In such amanner, in the third embodiment, the surface area of the support body 1is increased, and an amplified amount of the nucleic acids in one regionis increased. Then, as a result, the signal detectable from the whole ofthe nucleic acid colony is increased, and the measurement accuracy ofthe nucleic acid sequence can be enhanced.

Moreover, in the third embodiment; since the fibrous protrusions 23 aremade of silicon dioxide, the surfaces thereof are hydroxylated more ascompared with the surface of the substrate 19. Hence, bonding propertiesbetween the fibrous protrusions 23 and the chemical substance forbonding the nucleic acid templates and the colony primers are enhanced,and the nucleic acid templates and the colony primers can be fixed witha higher density.

Moreover, as shown in FIG. 16, the regions 20 to 22 where the fibrousprotrusions 23 are formed have high hydrophilicity as compared with theexposed surface (surface on which the fibrous protrusions 23 are notformed) of the substrate 19. Hence, in the case of supplying a polarsolvent onto the substrate 19, an occurrence of bubbles can besuppressed in the region where the nucleic acid templates T1 and thecolony primers X1 and X2 are formed since the polar solvent has highaffinity with the fibrous protrusions 23. Hence, the measurement canalso be formed with high accuracy.

Furthermore, in the third embodiment, the nucleic acid templates T1 areformed on the fibrous protrusions 23, and accordingly, in the regionwhere the nucleic acid templates T1 are fixed, reflectivity thereof islowered as compared with the flat surface. Hence, for example, in thecase of performing the analysis by fluorescence, excitation lightgenerated in the case of irradiating silicon atoms with light is mixedwith reflected light from the substrate 19, and sometimes becomes afactor of noise at the time of the fluorescence detection. However, inthe present invention, the above-described excitation light can bereduced by being shaded by the fibrous protrusions 23, and accordingly,the noise can be reduced as a result.

In the third embodiment, the plurality of fibrous protrusions 23 areformed on each of the regions 20 to 22; however, one fibrous protrusion23 may be formed on each of the regions 20 to 22. Also in this case, thesurface area of the substrate 19 is increased, and the nucleic acids canbe amplified more.

Moreover, in the third embodiment, the immobilized nucleic acids areamplified on the fibrous protrusions 23 by using the nucleic acidtemplates T1 as templates; however, other fixing methods of the nucleicacid strands may be adopted. For example, nucleic acids amplified inadvance may be flown on the fibrous protrusions 23, and the nucleicacids may be fixed by being individually bonded thereto. As describedabove, the sequencer of the third embodiment, which has the fibrousprotrusions 23, is used for fixing the variety of nucleic acids. Then,in each of such sequencers according to the respective modes, thesurface of the substrate 19 is increased by the fibrous protrusions 23,and accordingly, the number of nucleic acids which can be fixed in theunit region is increased, and the measurable signal is also increased.Hence, the measurement accuracy of the nucleic acid sequence can beenhanced.

Furthermore, in the third embodiment, as an example of the measurementmethod of the nucleic acid sequence, there is mentioned the method ofanalyzing the fluorescence by the hybridization between the fixednucleic acid strands (nucleic acid templates) and the nucleotideprecursors. However, this measurement method is merely an example, and avariety of methods can be used. For example, there is also a method ofmeasuring the sequence by capturing, as a change of light or an electricsignal, a state of an electromagnetic field changed by the hybridizationreaction between the fixed nucleic acid strands and the nucleotideprecursors.

(Fourth Embodiment)

A main difference between the fourth embodiment and the third embodimentis a shape of the substrate 19 as shown in FIG. 19. Specifically, in thefourth embodiment, recessed portions 28 are formed in advance on thesurface of the substrate 19 by wet etching or dry etching, and on bottomsurfaces of the recessed portions 28, the fibrous protrusions 23 areformed. Accordingly, in the fourth embodiment, the fibrous protrusions23 on the respective regions 20 to 22 can be suppressed from growingbeyond the regions 20 to 22. Specifically, a growing direction of thefibrous protrusions 23 is suppressed by suppressing surface diffusionthereof. Hence, shape accuracy of the regions where the fibrousprotrusions 23 are formed can be enhanced. Hence, each nucleic acidcolony can be formed for each of the regions 20 to 22, and the signalcan be measured with high accuracy. Moreover, in the fourth embodiment,for example, in the step of injecting the monomer solution onto thesubstrate 5 or injecting the measurement liquid thereonto, diffusion ofthe monomer solution or the measurement liquid can be prevented. Hence,the inspection sensitivity of the sensor is enhanced.

In the fourth embodiment, the fibrous protrusions 23 are formed on thebottom surfaces and side wall surfaces of the recessed portions 28;however, the fibrous protrusions 23 may be formed only on the bottomsurfaces of the recessed portions 28, or only on the sidewall surfacesthereof.

Note that a dent depth of the recessed portions 18 is preferably madedeeper than a thickness of layers where the fibrous protrusions 9 areformed since it becomes easy to suppress the diffusion. More preferably,the dent depth of the recessed portions 18 is made deeper, by severalten micrometers or more, than the thickness of the layers where thefibrous protrusions 9 are formed. Further, ranges of the recessedportions 18 can be appropriately selected in response to the purpose.

Descriptions of other configurations and effects, which are similar tothose of the first embodiment, will not be given, Description of otherconfigurations and effects, which are similar to those of the thirdembodiment, will not be given.

In each of the first to fourth embodiments, the flat substrate is usedas the base material; however, base materials with various shapes, forexample, such as a spherical shape and a cubic shape, may be used.

A sensor according to the present invention can be used as a DNA sensor.Moreover, besides the DNA sensor, the sensor according to the presentinvention can be used as a variety of sensors such as a protein sensor,a sugar sensor, and an antigen-antibody sensor. As the variety ofsensors for use, there are; a sensor that bonds, to surfaces of fibrousprotrusions, a receptor and a reactive substance, which are capable ofindividually capturing ligand molecules desired to be detected, andmeasures the bonding of the receptor and the reactive substance to theligand molecules; and a sensor that bonds, to the surfaces of thefibrous protrusions, ligand molecules desired to be measured asfunctional molecules, and reacts the ligand molecules with thesereceptor and reactive substance. If the functional molecules arepolymers, monomers can be polymerized in a predetermined region by usingblocking groups. Moreover, in a sequencer of the present invention, asequence of nucleic acids such as DNAs can be measured with highaccuracy.

The invention claimed is:
 1. A sensor comprising: a base material havinga surface; and fibrous protrusions formed from amorphous silicon dioxideand the fibrous protrusions being undulating and curling, having acrimped shape, and being configured so as to intertwine with otherfibrous protrusions, wherein each fibrous protrusion of the fibrousprotrusions has functional molecules formed respectively thereon.
 2. Thesensor according to claim 1, wherein the base material is formed fromsilicon, and the fibrous protrusions are directly joined to the surfaceof the base material.
 3. The sensor according to claim 1, wherein a partof the surface of the base material where the fibrous protrusions areformed has a hydrophilicity and water retention properties higher thanthe hydrophilicity and water retention properties of another part of thesurface where the fibrous protrusions are not formed.
 4. The sensoraccording to claim 1, wherein the base material has a recessed portionwith a bottom surface and a side wall surface, and the fibrousprotrusions are formed on at least one of the bottom surface and theside wall surface.
 5. The sensor according to claim 1, wherein eachfibrous protrusion of the fibrous protrusions has a plurality ofbranches.
 6. The sensor according to claim 1, wherein the surface of thebase material is partitioned into a plurality of regions, and thefibrous protrusions are formed on each of these regions.
 7. The sensoraccording to claim 1, wherein the fibrous protrusions have a pluralityof branches.